PROJECT SUMMARY/ABSTRACT Crohn’s disease is a debilitating and progressive inflammatory disease of the gastrointestinal tract. Defects in epithelial cells are thought to contribute to Crohn’s disease progression. However, the biological drivers and mechanisms of epithelial defects are not well understood. If epithelial cell defects occur secondary to the inflammatory and altered microbiome exposures present in the patient, treatments targeting these exposures would be predicted to resolve the epithelial defects. Alternatively, if epithelial defects are primary or durable contributors to Crohn’s disease progression, they would be predicted to persist despite treatment, as no current treatments target the epithelium. This project aims to distinguish these two models in order to improve Crohn’s disease prognosis. We have identified two quantitative epithelial defects related to microvillar dysfunction in Crohn’s disease ileal tissues lacking histological inflammation, suggesting that microvillar defects could be durable contributors to Crohn’s disease progression. Microvilli are apical membrane protrusions on epithelial cells that increase surface area for absorption and provide a physical location for the enrichment of enzymes, transporters, and host defense proteins critical for intestinal homeostasis. Loss of microvilli or certain microvillar localized proteins is associated with microvillus inclusion disease, very-early onset inflammatory bowel disease, and enteropathy. For this project, we propose to track the quantitative microvillar defects in vivo (pre- and post- treatment) and in vitro (epithelium removed from inflammatory/microbial exposures). We have developed an overarching hypothesis that epithelial microvillar defects will be durable in a Crohn’s disease patient subset with a more aggressive disease course. In Aim 1, we will investigate the durability of the microvillar defects in vivo using existing longitudinal samples and data from the pediatric RISK Crohn’s disease cohort and control subjects. We predict that the microvillar defects are more likely to persist in patients with progressive disease behavior, lack of anti-TNF response, and requirement for surgery. We will also perform association analysis with the microvillar defect phenotypes, clinical parameters, and microbiome profiles to identify ways we can potentially improve patient subsetting. In Aim 2, we will investigate the durability of the microvillar defects in vitro using human intestinal epithelial spheroid lines derived from Crohn’s disease and control patient biopsy tissues. Our preliminary analysis indicates that the microvillar defects will be durable in a subset of spheroid lines. We will test if durable spheroid microvillar defects are associated with decreased lipid metabolism, a critical enterocyte function predicted to be decreased by our preliminary analysis. Overall, this project will determine whether microvillar defects are durable epithelial contributors t...